Not applicable.
The present invention relates generally to Activity Dependent Neurotrophic Factor III (ADNF III), also known as Activity Dependent Neuroprotective Protein (ADNP). More particularly, the present invention relates to nucleic acid sequences encoding ADNF III polypeptides; ADNF III polypeptides encoded by such nucleic acid sequences; antibodies to ADNF III polypeptides; and methods of using such ADNF III polypeptides for the treatment of neurological deficiencies and for the prevention of cell death associated with (1) gp120, the envelope protein from HIV; (2) N-methyl-D-aspartic acid (excito-toxicity); (3) tetrodotoxin (blockage of electrical activity); and (4) xcex2-amyloid peptide, a substance related to neuronal degeneration in Alzheimer""s disease.
Neuronal division, survival and differentiation are dependent during development on a diverse group of protein and peptide growth factors. Included in this group of regulatory molecules are recognized trophic factors, such as nerve growth factor (NGF) (Levi-Montalcini, Differentiation, 13:51-53 (1979)), ciliary neurotrophic factor (CNTF) (Lin et al., Science 24:1023-1025 (1989)), fibroblast growth factor (FGF) (Wallicke et al., J. Neurosci. 11:2249-2258 (1991)), insulin-like growth factors 1 and 2 (IGFs 1 and 2) (Ishii et al., Pharmacol. and Ther. 62:125-144 (1994)), brain derived neurotrophic factor (BDNF) (Laibrock et al., Nature 341:149-152 (1989)), glial derived neurotrophic factor (GDNF) (Lin et al., Science 260:1130-1132 (1993)), and neurotrophin-3 and neurotrophin-4/5 (Henderson et al., Nature 363:266-269 (1993)). In addition, cytokines also have neurotrophic properties (Brenneman et al., J. Neurochem. 58:454-460 (1992); Patterson, Curr. Opin. Neurobiol. 2:91-97 (1992)). Although many of the classic growth factors were first recognized as playing important trophic roles in neuron/target cell interactions, it is now clear that glial cells in the central nervous system (CNS) express most of these growth factors/cytokines, and that these support cells play significant roles during development and nerve repair/regeneration.
In this regard, efforts have been made to understand the role of neuropeptides in regulating the release/expression of glia-derived trophic substances and to identify new glial molecules that contribute to the survival of developing CNS neurons. In particular, efforts have been made to understand the role of trophic support for activity-dependent neurons in the CNS. The activity-dependent neurons are a class of neurons that die during electrical blockade due to a reduction of soluble trophic materials in their environment (Brenneman et al., Dev. Brain Res. 9:13-27 (1993); Brenneman et al., Dev. Brain Res. 15:211-217 (1984)). Electrical blockade has been demonstrated to inhibit the synthesis and release of trophic materials into the extracellular milieu of CNS cultures (Agostan et al., Mol. Brain. Res. 10:235-240 (1991); Brenneman et al., Peptides 6(2):35-39 (1985)). Included in this trophic mixture is vasoactive intestinal peptide (VIP) (Brenneman et al., Peptides, supra (1985); Brenneman et al., Proc. Natl. Acad. Sci. USA 83:1159-1162 (1986)).
The 28-amino acid peptide VIP (Said et al., Ann. NY Acad. Sci. 527:1-691 (1988)), has been associated with cellular protection in sensory neurons, axotomized sympathetic neurons and acutely injured lung and airways (see, e.g., Gressens et al., J. Clin. Invest. 100:390-397 (1997)). Indeed, the lack of regulation of VIP expression observed in these injured or inflamed systems probably represents an adaptive response that limits damage and promotes recovery.
VIP has been shown to interact with high affinity receptors present on glial cells (Gozes et al., J. Pharmacol. Exp. Therap. 257:959-966 (1991)), resulting in the release of survival-promoting substances (Brenneman et al., J. Cell. Biol. 104:1603-1610 (1987); Brenneman et al., J. Neurosci. Res. 25:386-394 (1990)), among which are a glial-derived cytokine IL-1-xcex1 (Brenneman et al., J. Neurochem. 58:454-460 (1992); Brenneman et al., Int. J. Dev. Neurosci. 13:137-200 (1995)), and protease nexin I, a serine protease inhibitor (Festoff et al., J. Neurobiol. 30:255-26 (1995)). However, the neuronal survival-promoting effects of the VIP-conditioned medium were observed at very low concentrations that could not be attributed to IL-1 or protease nexin I released from astroglia. Therefore, efforts have been made to identify other survival-promoting proteins released from glial cells stimulated by VIP.
In doing so, a novel neuroprotective protein secreted by astroglial in the presence of VIP was discovered (Brenneman and Gozes, J. Clin. Invest. 97:2299-2307 (1996); Gozes and Brenneman, J. Molec. Neurosci. 7:235-244 (1996)). The neurotrophic protein was isolated by sequential chromatographic methods combining ion exchange, size separation and hydrophobic interaction. This neuroprotective protein (mol. mass, 14 kD and pI, 8.3xc2x10.25) was named Activity Dependent Neurotrophic Factor (ADNF or ADNF I) for two reasons: (1) a blockade of spontaneous electrical activity was necessary to detect the neuroprotective action of this substance in dissociated spinal cord cultures; and (2) VIP, a secretagogue for ADNF, was released during electrical activity, making the presence of ADNF in the extracellular milieu indirectly dependent on spontaneous activity. ADNF was found to exhibit neuroprotection at unprecedented concentrations. More particularly, femtomolar concentrations of ADNF were found to protect neurons from death associated with a broad range of toxins, including those related to Alzheimer""s disease, the human immunodeficiency virus (HIV), excitotoxicity, and electrical blockade (see, e.g., Gozes et al., Dev. Brain Res. 99:167-175 (1997)).
During the course of studies directed to the structural characteristics of ADNF, an active peptide fragment of ADNF was discovered. This active peptide, 9-amino acids derived from ADNF (ADNF-9), was found to have strong homology, but not identity, to an intracellular stress protein: heat shock protein 60 (hsp60). Another peptide, ADNF-14, which comprises ADNF-9, was also found to be active, as were other derivatives of ADNF-9. Moreover, ADNF-9 was shown to mimic the potency of the parent protein, while exhibiting a broader range of effective concentrations as compared to the parent protein. In addition, ADNF-9, like ADNF, has been shown to prevent neuronal cell death associated with the envelope protein (gp120) from HIV (see Dibbern et al., J. Clin. Invest. 99:2837-2841 (1997)), with excitotoxicity (N-methyl-D-aspartate), with the xcex2-amyloid peptide (putative cytotoxin in Alzheimer""s disease), and with tetrodotoxin (electrical blockade) (see Brenneman and Gozes, J. Clin. Invest. 97:2299-2307 (1996)).
The discovery of ADNF has provided additional knowledge regarding the neuroprotective action of VIP (Gozes and Brenneman, Mol. Neurobiol. 3:201-236 (1989); Said, J. Clin Invest. 97:2163-2164 (1996)). Moreover, the neurotrophic properties of the ADNF polypeptide have significant therapeutic and diagnostic implications. The discovery that ADNF activity can be mimicked by a 9-amino acid peptide is predicted to facilitate innovative drug design for the treatment of the neurological symptoms associated with HIV infection, Alzheimer""s disease, and other prevalent neuro-degenerative diseases. Although ADNF, ADNF-9, and ADNF-14 have unlimited potential as neuroprotectants, it would still be advantageous to identify other survival-promoting proteins released from glial cells stimulated by VIP.
The present invention relates to the discovery of a nucleic acid encoding a novel neuroprotective polypeptide, i.e., Activity Dependent Neurotrophic Factor III (ADNF III), also called Activity Dependent Neuroprotective Protein (ADNP). As with the previously described ADNF I, ADNF III exhibits potent neuroprotective effects, with the EC50 of such neuroprotective effects being in the femtomolar range. Based on the recognized homology between ADNF I and hsp60, a heat shock protein, and PIF1, a DNA repair protein, these two epitopes were utilized to prepare antibodies which, in turn, were used to screen a mouse cDNA-expression library to identify the new neuroprotective polypeptide ADNF III. One mouse ADNF III cDNA clone consists of about 2418 base pairs of an open reading frame, which encodes an ADNF III polypeptide of about 806 amino acids, pI 5.85 (nucleotide sequence, SEQ ID NO:4; amino acid sequence, SEQ ID NO:3, see FIG. 1). An additional mouse cDNA has been cloned, encoding an ADNF III polypeptide of about 828 amino acids, pI 5.99 (nucleotide sequence, SEQ ID NO:54; amino acid sequence, SEQ ID NO:55; see FIG. 11). Human cDNAs encoding ADNF III have also been cloned (xe2x80x9cH3xe2x80x2xe2x80x9d nucleotide sequence, SEQ ID NO:2; xe2x80x9cH3xe2x80x2xe2x80x9d amino acid sequence, SEQ ID NO:1; xe2x80x9cH3xe2x80x9d nucleotide sequence, SEQ ID NO:56 and FIG. 12; xe2x80x9cH3xe2x80x9d amino acid sequence, SEQ ID NO:57 and FIG. 12; xe2x80x9cH7xe2x80x9d nucleotide sequence, SEQ ID NO:58 and FIG. 13; and xe2x80x9cH7xe2x80x9d amino acid sequence, SEQ ID NO:59 and FIG. 13). The mouse and human cDNAs demonstrate about 88.7% homology at the nucleotide level (compare SEQ ID NOS:54 and 58). The promoter sequence for ADNF III has also been cloned (SEQ ID NO:60 and FIG. 14).
Based on the homology between ADNF I and hsp60 to ADNF III, an eight-mer ADNF III polypeptide was synthesized that exhibited structural homology to hsp60 and to the previously described ADNF-9 active peptide SALLRSIPA (SEQ ID NO:5). This ADNF III polypeptide is 8 amino acids in length and has the sequence NAPVSIPQ, i.e., Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:6). Both the xe2x80x9cexpressedxe2x80x9d (full length) ADNF III polypeptides and NAPVSIPQ-derived (SEQ ID NO:6) ADNF III polypeptides of the present invention are extraordinarily potent in preventing neuronal cell death. Such ADNF III polypeptides have been found to exhibit neuroprotection against neurotoxins associated with HIV infection, electrical blockage, excitotoxicity and Alzheimer""s disease.
As such, in one embodiment, the present invention provides isolated nucleic acids encoding the ADNF III polypeptides of the present invention, the ADNF III polypeptides including, for example, those that specifically bind to antibodies generated against immunogens having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:55, SEQ ID NO:57, and SEQ ID NO:59, and conservatively modified variants thereof. The ADNF III nucleic acids of the present invention also include those encoding amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:55, SEQ ID NO:57, and SEQ ID NO:59, and conservatively modified variants thereof. Exemplar nucleic acids include those set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:58. Other nucleic acids encoding the ADNF III polypeptides of the present invention include those with silent codon substitutions relative to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, as well as conservatively modified variations thereof.
Isolated nucleic acids that specifically hybridize, under stringent conditions, to the exemplar nucleic acids, i.e., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, are also provided. For example, a complementary nucleic acid to a sequence provided by SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:54, SEQ ID NO:56, or SEQ ID NO:58 specifically hybridizes to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:54, SEQ ID NO:56, or SEQ ID NO:58, respectively. Similarly, nucleic acids that have substantial subsequence complementary to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:54, SEQ ID NO:56, or SEQ ID NO:58 also specifically hybridize to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:54, SEQ ID NO:56, or SEQ ID NO:58, respectively. Still other nucleic acids encoding the ADNF III polypeptides of the present invention include those that are amplified by primers that specifically hybridize under stringent hybridization conditions to the same sequence as a primer set selected from the group consisting of: sense 5xe2x80x2 TCCAATGTTCACCTGCAG 3xe2x80x2 (SEQ ID NO:7), sense 5xe2x80x2 ACCTGCAGCAAAACAACTAT 3xe2x80x2 (SEQ ID NO:9), and antisense 5xe2x80x2 GCTCGTTACAGATTGTAC 3xe2x80x2 (SEQ ID NO:8).
Isolated nucleic acids that specifically hybridize, under stringent conditions, to the exemplar promoter nucleic acids, i.e., SEQ ID NO:60, are also provided. For example, a complementary nucleic acid to a sequence provided by SEQ ID NO:60 specifically hybridizes to SEQ ID NO:60. Similarly, nucleic acids that have substantial subsequence complementary to SEQ ID NO:60 also specifically hybridize to SEQ ID NO:60.
In a presently preferred embodiment, the isolated nucleic acids of the present invention are optionally vector nucleic acids, which comprise a transcription cassette. More particularly, the vectors preferably include the above-described nucleic acids operably linked (under the control of) a promoter; either constitutive or inducible. The promoter may be heterologous or may be an ADNF III promoter. The vector can also include initiation and termination codons. The transcription cassette optionally encodes a polypeptide. Typically, the portion of the transcription cassette that encodes the polypeptide specifically hybridizes, under stringent conditions, to a nucleic acid selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:54, SEQ ID NO:56, or SEQ ID NO:58. The promoter region may also hybridized under stringent conditions to a nucleic acid having the sequence of SEQ ID NO:60. Upon transduction of the transcription cassette into a cell, an mRNA is produced that specifically hybridizes, under stringent conditions, to a nucleic acid selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:54, SEQ ID NO:56, or SEQ ID NO:58. The mRNA is translated in the cell into an ADNF III polypeptide, such as the ADNF III polypeptides comprising amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:55, SEQ ID NO:57, and SEQ ID NO:59, and conservatively modified variants thereof.
In another embodiment, the present invention provides ADNF III polypeptides. Such ADNF III polypeptides include those encoded by nucleic acids that specifically hybridize, under stringent conditions, to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:54, SEQ ID NO:56, or SEQ ID NO:58. Such ADNF III polypeptides also include those that specifically bind an antibody generated against an immunogen having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:55, SEQ ID NO:57, and SEQ ID NO:59 and conservatively modified variations thereof. Exemplar ADNF III polypeptides include ADNF III polypeptides having the amino acid sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:55, SEQ ID NO:57, and SEQ ID NO:59 and conservatively modified variations thereof.
In yet another embodiment, the ADNF III polypeptides of the present invention comprise the following amino acid sequence:
(R1)x-Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln-(R2)y (SEQ ID NO:10)
and conservatively modified variations thereof. In the above formula, R1 is an amino acid sequence comprising from 1 to about 40 amino acids, wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs. The term xe2x80x9cindependently selectedxe2x80x9d is used herein to indicate that the amino acids making up the amino acid sequence R1 may be identical or different (e.g., all of the amino acids in the amino acid sequence may be threonine, etc.). Moreover, as previously explained, the amino acids making up the amino acid sequence R1 may be either naturally occurring amino acids, or known analogues of natural amino acids that functions in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics and analogs). Suitable amino acids that can be used to form the amino acid sequence R1 include, but are not limited to, those listed in Table I, supra. The indexes xe2x80x9cxxe2x80x9d and xe2x80x9cyxe2x80x9d are independently selected and can be equal to one or zero.
As with R1, R2, in the above formula, is an amino acid sequence comprising from 1 to about 40 amino acids, wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs. Moreover, as with R1, the amino acids making up the amino acid sequence R2 may be identical or different, and may be either naturally occurring amino acids, or known analogues of natural amino acids that functions in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics and analogs). Suitable amino acids that can be used to form R2 include, but are not limited to, those listed in Table I, supra.
In a further embodiment, the present invention provides antibodies that specifically bind to ADNF III polypeptides. In a preferred embodiment, the antibodies specifically bind to an ADNF III polypeptide, the ADNF III polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:55, SEQ ID NO:57, and SEQ ID NO:59 and conservatively modified variations thereof.
Quite surprisingly, it has been discovered that the ADNF III polypeptide of the present invention can be used for the treatment of neurological deficiencies and for the prevention of neuronal cell death. Such ADNF III polypeptides can be used, for example, to prevent the death of neuronal cells including, but not limited to, spinal cord neurons, hippocampal neurons, cerebral neurons and cholingeric neurons. More particularly, the ADNF III polypeptides of the present invention can be used to prevent cell death associated with (1) gp120, the envelope protein from HIV; (2) N-methyl-D-aspartic acid (excito-toxicity); (3) tetrodotoxin (blockage of electrical activity); and (4) xcex2-amyloid peptide, a substance related to neuronal degeneration in Alzheimer""s disease.
As such, the present invention provides methods for preventing neuronal cell death. More particularly, in one aspect, methods are provided for using the ADNF III polypeptides of the present invention to prevent gp120-induced neuronal cell death in a patient infected with HIV. In another aspect, methods are provided for using the ADNF III polypeptides of the present invention to prevent neuronal cell death associated with excito-toxicity induced by N-methyl-D-aspartate stimulation. In yet another aspect, methods are provided for using the ADNF III polypeptides of the present invention to prevent neuronal cell death induced by the xcex2-amyloid peptide in a patient afflicted or impaired with Alzheimer""s disease. In still another aspect, methods are provided for using the ADNF III polypeptides of the present invention to alleviate learning impairment produced by cholingeric blockage in a patient impaired or afflicted with Alzheimer""s disease.
In addition to the foregoing, the ADNF III polypeptides of the prevent invention can effectively be used to prevent neuronal cell death associated with a number of other neurological diseases and deficiencies. More particularly, as a result of their ability to inhibit neuronal cell death associated with N-methyl-D-aspartic acid (excitotoxicity), the ADNF III polypeptides of the present invention can be used to treat numerous forms of neurodegeneration (see Lipton and Rosenberg, New Eng. J. Med. 330:613-622 (1994), the teaching of which are incorporated herein by reference for all purposes). Such neurodegeneration includes, but is not limited to, the following: Huntington""s disease; AIDS dementia complex; epilepsy, neuropathic pain syndromes; olivopontocerebellar atrophy; parkinsonism and Parkinson""s disease; amyotrophic lateral sclerosis; mitochondrial abnormalities and other inherited or acquired biochemical disorders; MELAS syndrome; MERRF; Leber""s disease; Wernicke""s encephalopathy; Rett syndrome; homocysteinuria; hyperprolinemia; nonketotic hyperglycinemia; hydroxybutyric aminoaciduria; sulfite oxide deficiency; combined systems disease; lead encephalopathy; Alzheimer""s disease; hepatic encephalopathy; Tourette""s syndrome; oxidative stress induced neuronal death; Down""s syndrome; developmental retardation and learning impairments; closed head trauma; dompamine toxicity; drug addiction, tolerance, and dependency. Those of skill in the art will appreciate that the above list is illustrative and not exhaustive, and that the ADNF III polyepeptides of the present invention can be used to treat other neurological disorders.
Other features, objects and advantages of the invention and its preferred embodiments will become apparent from the detailed description that follows.